Irreversible immobilization of enzymes into polyurethane coatings

ABSTRACT

A method of irreversibly immobilizing an enzyme in a polyurethane and an enzyme-containing polyurethane having a degree of immobilization of the enzyme of approximately 100%. The synthesis of waterborne polyurethanes in the presence of enzyme has enabled the irreversible attachment of the enzyme to the polymeric matrix. The distribution of immobilized enzyme as well as activity retention are homogeneous within the polyurethane. Decreasing ECC hydrophobicity, via the use of a less hydrophobic polyisocyanate prepolymer during polymerization, significantly enhanced the intrinsic activity of the ECC.

The present invention is a divisional of pending U.S. application Ser.No. 10/850,674 entitled “Irreversible Immobilization of Enzymes intoPolyurethane Coatings” filed 21 May 2004 and assigned to the sameassignee as the present invention, which is a continuation in partapplication based on U.S. Ser. No. 10/202,224 filed on Jul. 24, 2002,which application claimed the benefit of 35 U.S.C. § 119(c) ofprovisional application Ser. No. 60/307,450 entitled “IrreversibleImmobilization Of Diisopropylfluorophosphatase Into PolyurethaneCoatings” filed on Jul. 24, 2001 which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the irreversible immobilization ofenzymes into polyurethanes, particularly, polyurethane coatings,adhesives, and sealants.

2. State of the Art

Immobilization has been widely employed to enable and enlarge theapplication of enzymes as catalysts in industrial processes.Polyurethane foam has been employed as polymeric support for bioplasticsynthesis with various enzymes over the last decade. Polyurethanesponge-like polymers may be synthesized from hydrophilic toluenediisocyanate (TDI)- or methylene bis (ρ-phenylisocyanate) (MDI)-basedpolyisocyanate prepolymers and water. The incorporation of enzymes inmonolithic polyurethane foam is often characterized by a degree ofimmobilization close to 100% and a high activity retention.Thermostability enhancement via immobilization in polyurethane foams hasalso been reported.

The insertion of biological molecules in coatings and thin films maydrive a large range of applications. For example, potentiometricbiosensors often involve the covalent attachment of enzyme onto an innerfilm adjacent to the sensing surface of the electrode, and thesubsequent protection of the enzyme layer with an outer film. Anotherimmobilization method for the fabrication of amperometric biosensorsrelies on the entrapment of enzyme in a gel layer, which is furthercoated by an external protective film. The lifetime and use of suchsystems are often limited by the diffusion of enzyme through theexternal membrane. To overcome this main disadvantage, the enzyme has tobe directly and covalently immobilized into the coating. The covalentincorporation of biocatalyst into coatings would also be beneficial forother bioprocesses such as biocatalytic separation and filtration,microchips, and antifouling.

Direct covalent immobilization of highly-active enzymes into coatingsand films has remained an elusive goal, with some of the most successfulapproaches exhibiting only up to 0.5% activity. Waterborne polyurethanecoatings result from the polymerization of an aqueous polyol with awater dispersible polyisocyanate. As the film is cured at roomtemperature, water evaporates and cross-linking occurs through thecondensation between hydroxyl groups and isocyanate functionalities.Two-component waterborne polyurethanes are increasingly used inindustrial applications, as they exhibit properties similar to those ofsolvent borne polyurethane coatings. Waterborne polyurethane coatingrepresents a potentially ideal polymeric matrix for multipoint andcovalent immobilization of enzymes.

In view of the foregoing, there is a need in the art for a method bywhich an enzyme can be directly added to the aqueous phase of atwo-component system prior to polymerization. The immobilization processrelies on the ability of amines at the enzyme surface to react withisocyanate functionalities at a faster rate than hydroxyl groups on theprepolymer.

SUMMARY OF THE INVENTION

The present invention includes a method of irreversibly immobilizingenzyme into polyurethane materials comprising the steps of: reacting amixture of one or more polyol dispersion coreactant(s) and one or moreenzyme(s) to create an aqueous mixture; adding one or morewater-dispersible polyisocyanate(s) to the aqueous mixture in an amountsufficient to produce a dispersion capable of forming a polyurethanematerial; applying the dispersion onto one or more substrates to createan enzyme-containing material; and curing the enzyme-containingmaterial. This method is particularly useful for the production ofenzyme-containing polyurethane coatings, adhesives and sealants.

Additionally, the present invention includes a method of irreversiblyimmobilizing diisopropylflurophosphatase into polyurethanes,particularly, polyurethane coatings, adhesives and sealants comprisingthe steps of: reacting a mixture of a polyol dispersion coreactant, withthe dispersion preferably having a water content of from about 10 toabout 90 wt. %, a polyether modified polydimethyl siloxane surfactant, abuffered medium, preferably comprising bis-tris propane buffer and CaCl₂and diisopropylflurophosphatase to create an aqueous mixture; adding awater-dispersible polyisocyanate, preferably, a polyisocyanate based onhexamethylene diisocyanate to the aqueous mixture to produce adispersion or an emulsion; applying the dispersion or emulsion onto asubstrate to create an enzyme-containing material, preferably, acoating, adhesive or sealant; and curing the enzyme-containing materialwhich is preferably a coating, adhesive or sealant.

Also, the present invention includes an enzyme-containing coating,adhesive or sealant made by the process comprising the steps of:reacting a mixture of a polyol dispersion coreactant, and an enzyme tocreate an aqueous mixture; adding a water-dispersible polyisocyanatebased on hexamethylene diisocyanate to the aqueous mixture and reactingto produce a dispersion or an emulsion; applying the dispersion oremulsion onto a substrate to create an enzyme-containing coating,adhesive or sealant; and curing the enzyme-containing coating, adhesiveor sealant for approximately 12 hours under ambient conditions.

These and other advantages and benefits of the present invention will beapparent from the Detailed Description of the Preferred Embodimentherein below.

BRIEF DESCRIPTION OF THE DRAWINGS

For the present invention to be readily understood and practiced, theinvention will now be described, for purposes of illustration and notlimitation, in conjunction with the following figures wherein:

Table 1 illustrates the kinetic parameters for DFPase-containingcoatings and soluble DFPase.

FIG. 1 illustrates a schematic of the DFP concentration profile in thecase of simultaneous diffusion and enzymatic reaction in theDFPase-containing polyurethane coating.

FIG. 2 illustrates an enzyme distribution in polyurethane coating.

FIG. 3 illustrates the effect of DFPase concentration onDFPase-containing coating efficiency.

FIG. 4 illustrates the effective diffusion of DFP through coatings.

FIG. 5 illustrates profiles for DFP consumption in diffusion cells.Coatings were synthesized using the polyol XP-7093 and polyisocyanateXP-7007, and a DFPase loading of 3.6 mg/g_(coating).

FIGS. 6 a and 6 b illustrates profiles for DFP consumption in diffusioncells.

FIG. 7 illustrates the thermoinactivation of DFPase-containing coatingat 65° C.

FIG. 8 illustrates the thermoactivation of DFPase-containing coating atroom temperature.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention relates to the immobilization of enzymes intowaterborne polyurethane materials such as coatings, adhesives andsealants. One such enzyme which may be used isdiisopropylflurophosphatase (DFPase, E.C. 3.8.2.1). However, thoseskilled in the art will recognize that a wide variety of enzymes andantibodies may be used. It is understood that native DFPase catalyzesthe hydrolysis of toxic organophosporus nerve agents such as soman anddiisopropylfluorophosphate (DFP). In the prior art, DFPase has beencopolymerized into monolithic polyurethane foams with a 67% activityretention and an enhanced thermostability. Since alterations inenzyme-containing coating (ECC) hydrophilicity could influence theactivity retention and stability, the immobilization process of thepresent invention was performed using polyisocyanate prepolymers withvarious hydrophilicities. The degree to which the enzyme wasirreversibly attached to the support was determined. The enzymedistribution within the coating was observed by means of gold-labeling.The influence of mass transfer on the activity of enzyme-polymers wasexamined using a diffusion cell apparatus. The enhancement of DFPasethermostability via immobilization was also investigated.

It is well known that waterborne polyurethane coatings, adhesives andsealants may be produced by reacting a polyol dispersion or a mixture ofpolyol dispersions with a water dispersible polyisocyanate or mixture ofpolyisocyanates. Such known processes and the materials suitable forsuch processes include those disclosed in, for example, U.S. Pat. Nos.4,663,377; 5,075,370; 5,098,983; 5,389,718; and 5,200,489 which areincorporated herein by reference.

Materials and Methods

Material

Any of the known polyisocyanates which may be sufficiently dispersed inwater to produce a coating, adhesive or sealant may be used in thepractice of the present invention. Water dispersible aliphaticpolyisocyanates such as BAYHYDUR polyisocyanates XP-7063, XP-7007,XP-7148 and Desmodur N3400 are particularly suitable. Any of the polyolsor polyol dispersions known to be useful for producing coatings,adhesives and/or sealants may be used in the practice of the presentinvention. Polyol dispersions such as BAYHYDROL polyol XP-7093 areparticularly preferred. As used in the present application, the termsdispersion and emulsion are used interchangeably. The substrate to whichthe dispersion is applied may be composed of any material to which thepolyurethane will adhere. Suitable substrates include wood, steel,glass, concrete and plastics. Thermoplastic polyolefin (TPO) panels areparticularly preferred. The polyisocyanates, polyols and TPO panels usedin the synthesis and curing of protein-containing coatings describedmore fully below were kindly provided by Bayer Co. (Pittsburgh, Pa.).BAYHYDUR polyisocyanates XP-7063, XP-7007, XP-7148 and Desmodur N3400are water dispersible aliphatic polyisocyanates based on hexamethylenediisocyanate (HDI). BAYHYDROL polyol XP-7093 is a polyol dispersion. Thesurfactant BYK-345, which is a polyether modified polydimethyl siloxane,was obtained from BYK-Chemie (Wallingford, Conn.).Diisoproplyfluorophosphate (DFP), Bradford reagent, bovine serum albumin(BSA), Bis-Tris Propane, Tris(hydroxylmethyl)aminomethane HCl(Tris-HCl), CaCl₂, NaCl, K₂CO₃ and isopropanol were purchased fromSigma-Aldrich Chemical Co. (St. Louis, Mo.). DFPase was purchased fromBioCatalytics, Inc. (Pasadena, Calif.). Polybed 812 embedding resin,which is an epoxy resin, was obtained from Polysciences (Warrington,Pa.).

Method

ECC Synthesis

ECC's were prepared using buffered aqueous mixtures (10 mMBis-Tris-Propane buffer, pH 7.5, 5 mM CaCl₂). Waterborne two-componentpolyurethanes were synthesized using water-dispersible aliphaticpolyisocyanates based on hexamethylene diisocyanate (HDI) BAYHYDUR andpolyol dispersion coreactants BAYHYDROL. During ECC synthesis, a ratiobetween isocyanate and hydroxyl functionalities of 2 was used.Typically, BAYHYDROL XP-7093 (2.5 g) (water content of 70 wt. %),BYK-345 surfactant (0.1 g) and buffered medium (1.2 g) were poured intoa cylindrical vessel, and followed by the addition of enzyme, DFPase(0.02-9 mg). The aqueous solution was further stirred mechanically (300rpm) for 1 min. The amounts of BAYHYDUR XP-7063, XP-7007, XP-7148required for ECC synthesis were calculated knowing the polyisocyanateequivalent molecular weights. When using XP-7007, the polyisocyanate (1g) was added to the aqueous solution, and the biphasic mixture wasagitated for 20 s with a custom designed head attached to a 2500 rpmhand held drill. After mixing, a white emulsion with a 63 w % watercontent was obtained, and applied (0.45 g) on thermoplastic polyolefin(TPO) panels previously cleaned with isopropanol and dried under ambientconditions. The ECC was then allowed to cure for 12 hrs under ambientconditions and weighed again (0.24 g).

Bis-Tris-Propane contains hydroxyl groups and secondary amines, whichmight react with the isocyanates during the coating synthesis. Theamount of buffer salt added to the reaction mixture was negligible ascompared with the reactive functionalities of the polyisocyanate andpolyol dispersion, and, hence, did not appear to affect the propertiesof the resulting two-component waterborne polyurethanes.

Protein Concentration Determination

Protein concentrations were evaluated using the Bradford reagent. Theaddition of the dye to protein solution at room temperature results inthe formation of a dye-protein complex within fifteen (15) minutes, withan absorption maximum at 596 nm. A calibration curve with an extinctioncoefficient of 0.0341 ml/mg is obtained for protein concentrationsranging from 1 to 10 mg/ml.

Synthesis of Enzyme/Gold Conjugates

Gold colloids with diameters ranging from 25 to 30 nm were prepared andconjugated to DFPase in aqueous medium. Specifically, a gold solution(100 ml of 0.01% HAuCl₄.2H₂O) was heated in a glass flask until boiling.Trisodium citrate (5 ml at 0.015%) was added and the mixture was furtherboiled. The colloid formation was completed when a persistent orange/redcolor was obtained. During conjugation the pH was adjusted slightlyabove the enzyme isoelectric point (pI 5.8) with K₂CO₃. The pH wasmeasured with litmus paper. Typically, an enzyme weight of 0.12 g wasneeded to stabilize 30 ml of gold colloid solution (gold concentration:0.01%). After addition of DFPase, the enzyme-gold solution was gentlyagitated, and bovine serum albumin solution (BSA) (10% (w/v)) was addedto a final concentration of 0.1% (w/v). BSA blocked areas of thecolloidal surface that were not coated with the enzyme. The resultingsolution was centrifuged for 1 hr. at 100,000 rpm, and the enzyme-goldconjugate was recovered in the precipitate, which was resolubilized inbuffered medium (10 mM Tris-HCl, pH 7.5). Centrifugation led, to acertain extent, to the formation of gold clusters. The largest clustersthat were found in dense areas of the precipitate were discarded.Smaller clusters were still present among the colloidal gold conjugates.Coatings were further prepared with BAYHYDUR XP-7007 as described aboveusing two different concentrations of colloidal gold conjugated toenzyme (0.001 mg_(gold)/g_(coating) and 0.012 mg_(gold)/g_(coating)).

Localization of Gold-DFPase Conjugate in Coating

To embed the films for transmission electron microscopy (TEM), smallstrips were washed several times in 100% ethanol then incubated inseveral 1 hr. changes of Polybed 812 embedding resin. It should beunderstood that several embedding media may be used. Most of theembedding media which may be used are based on epoxy resin and modifiedepoxy resin or methacrylic polymers. Films were cut into 1 mm×2 mmstrips, placed in embedding molds and embedded in Polybed 812. Blockswere cured overnight at 37° C., then cured for two days at 65° C.Ultrathin cross sections (60 mm) of the films were obtained on aReichart Ultracut E microtome. Sections were viewed on a JEOL JEM 1210or 100CX transmission electron microscope at 80 KV.

Activity of ECC's Using a Fluoride Ion Electrode

ECC was assayed using pieces of peeled DFPase-film ranging in weightfrom 0.009 to 0.012 g. Typically, the pieces were placed in 10 ml of 3mM DFP buffered solutions (5 mM CaCl₂ and 10 nM Bis-Tris-Propane, pH7.5) and agitated by magnetic stirring. As DFPase acts by binding andhydrolyzing DFP (see below), the activity was measured by followingfluoride release with a fluoride ion electrode at room temperature.Fluoride bulk solution concentration was measured every 20 s for 5 min.

The enzyme concentration in the coatings was varied between 0 and 2mg/g_(coating). The ECC's with higher enzyme concentrations were tooactive for the initial velocities to be determined.

Determination of Kinetic Constants Using a Fluoride Ion Electrode

The kinetic constants were determined by means of a fluoride sensor asdescribed in the previous section. The substrate concentrations variedfrom 0 to 20 mM. The data were fit to the Michaelis-Menten equationusing a non-linear regression (Sigma Plot Version 2).

Diffusion Cell Experiments

The diffusion apparatus was composed of a donor and a receptorcompartment, each of them being equipped with a water jacket. Thediffusion system was composed of two horizontal side by side chamberswith defined compartment volume (3 ml) and diffusion cross-section area(ID=9 mm). The ECC was mounted between the two compartments, and theexperiments were conducted at room temperature (22° C.).

Determination of Substrate Effective Diffusion Coefficient, D_(eff)

The substrate effective diffusion coefficient, D_(eff)(m²/min), wasestimated by following the procedure, known in the art. Urease Type III(EC. 3.5.1.5), from Jack beans, which is commercially available fromSigma (St. Louis, Mo.) was immobilized into the coating (3.6mg/g_(coating)) to mimic the presence of DFPase. Initially, a 3 mlvolume of buffered medium (5 mM CaCl₂, 10 mM Bis-Tris-Propane, pH 7.5)supplemented with DFP (4 mM) was placed in the donor cell, while thereceptor cell was filled with buffered medium (3 ml). Each cell was wellmixed by magnetic stirring. After a fixed period of time (5-300 min),the contents were removed and diluted 4 times with buffer medium (5 mMCaCl₂, 10 mM Bis-Tris-Propane, pH 7.5). The DFP concentration of eachsample was then determined by an activity assay with soluble DFPase.D_(eff) was calculated at quasi-steady state (Equation 1).$\begin{matrix}{\lbrack{DFP}\rbrack_{R} = {\frac{D_{eff}{A\lbrack{DFP}\rbrack}_{D}}{V_{cell}\delta^{\prime}}\left( {t - t_{0}} \right)}} & {{Equation}\quad 1}\end{matrix}$[DFP]_(D) and [DFP}_(R) are the DFP concentrations in the donor andreceptor cell, respectively (mol/m³). V_(cell) (3.10⁻⁶ m³) and A(6.36.10⁻⁵ m²) are the cell volume and diffusion cross-section area,respectively. Assuming that the swelling of polyurethane film occurspredominantly in thickness, the thickness of wetted ECC, δ′, wasestimated as follows: $\begin{matrix}{\delta^{\prime} = {\frac{1}{1 - ɛ}\delta}} & {{Equation}\quad 2}\end{matrix}$The dry coating thickness, δ (10 μm), was determined using scanningelectron microscopy. ε (0.7) is the fraction of the total volumeoccupied by the liquid phase in the wetted coating.Activity Measurements

The cells were filled with buffer (5 mM CaCl₂, 10 mM Bis-Tris-Propane,pH 7.5). The donor cell was initially supplemented with DFP (4 mM). Theinitial DFP concentration in receptor cell was either 0 or 4 mM. Theexperiments were conducted using a fixed DFPase-ECC concentration (3.6mg/g_(coating)), for which the complete degradation of the substrateoccurred on a reasonable time scale. Each cell was well mixed bymagnetic stirring. After a fixed period of time (5-120 min), thecontents were removed and diluted 4 times with buffer (5 mM CaCl₂, 10 mMBis-Tris-Propane, pH 7.5). The DFP concentration of each sample was thendetermined by an activity assay with soluble DFPase.

FIG. 1 is a schematic of the DFP concentration profile in the case ofsimultaneous diffusion and enzymatic reaction in the DFPase-containingcoating when the receptor cell does not contain DFP at t=0 sec. 1 s, δare the stagnant solution layer and the coating thickness, respectively,C_(DFP,D,t), C_(DFP,R,t) are the bulk DFP concentrations at a time t inthe donor and receptor cell, respectively. C_(DFP,0,t), C_(DFP,δ,t) arethe DFP concentration in the liquid phase of coating at the surfaces andat a time. If the diffusional resistance of boundary layer and the ECCswelling is neglected, the concentration profiles of DFP in theDFPase-ECC at unsteady state are given by Equation 3. $\begin{matrix}{\frac{\mathbb{d}\lbrack{DFP}\rbrack_{lc}}{\mathbb{d}t} = {{D_{eff}\frac{\mathbb{d}^{2}\lbrack{DFP}\rbrack_{lc}}{\mathbb{d}^{2}x}} - \frac{{{k_{{cat}.{int}}\lbrack{DFPase}\rbrack}_{lc}\lbrack{DFP}\rbrack}_{lc}}{K_{M,{int}} + \lbrack{DFP}\rbrack_{lc}}}} & {{Equation}\quad 3}\end{matrix}$[DFP]_(lc) (mol/m³) is the DFP concentration in the liquid phase in thecoating, k_(cat,int) (s⁻¹) and K_(M) (mol/m³) are the intrinsic kineticconstants for the ECC.The initial conditions are as follows:x=0 and t=0, [DFP]_(lc)=4  Equation 4x(x≠0) and t=0, [DFP]_(lc)=0  Equation 5At the interface between the ECC and the donor cell we have:$\begin{matrix}{\frac{\mathbb{d}\lbrack{DFP}\rbrack_{0}}{\mathbb{d}t} = {{\frac{{AD}_{eff}}{V_{cell}}\frac{\mathbb{d}\lbrack{DFP}\rbrack}{\mathbb{d}x}}❘_{0}{- \left( {V_{Surface} + V_{Release}} \right)}}} & {{Equation}\quad 6}\end{matrix}$

Where [DFP]₀ represents the DFP concentration in the liquid phase at thesurface of the ECC (X=0). V_(surface) (mol/m³.s)) represents the rate ofDFP hydrolysis at the coating surface (x=0) (Equation 7), andV_(Release) (mol/(m³.s)) the rate of reaction catalyzed by the enzymenot covalently immobilized during the ECC synthesis and released in thedonor cell (Equation 8). $\begin{matrix}{V_{Surface} = \frac{{{k_{{cat},{int}}\lbrack{DFPase}\rbrack}_{Surface}\lbrack{DFP}\rbrack}_{0}}{K_{M,{int}} + \lbrack{DFP}\rbrack_{0}}} & {{Equation}\quad 7}\end{matrix}$

Where [DFPase]_(Surface) (mol/m³) is the number of moles of enzyme atthe coating surface per unit volume of donor cell. $\begin{matrix}{V_{Release} = \frac{{{k_{{cat},{native}}\lbrack{DFPase}\rbrack}_{Release}\lbrack{DFP}\rbrack}_{0}}{K_{M,{native}} + \lbrack{DFP}\rbrack_{0}}} & {{Equation}\quad 8}\end{matrix}$

Where [DFPase}_(Release) (mol/m³) is calculated with respect to thedonor cell volume, k_(cat,native) and K_(M,native) are given in Table 1(Experiment 1^(a*)).

Given the experimental DFP concentration profiles in donor and receptorcells Equation 2 was solved numerically using Equation 3 through 7 withAthena Visual Version 7.1.1. The intrinsic kinetic constants of the ECC,K_(M,int) and k_(catk,int) were then calculated.

Enzyme Modification with Desmodur N3400 Polyisocyanate

DFPase-containing solution (1 ml) (50 mM MOPS, 5 mM CaCl₂, pH 7.5) wasadded to Desmodur N3400 (1 g), which is composed of the dimer and trimerof HDI. The biphasic mixture was stirred at room temperature. Theactivity of modified enzyme was determined by means of a fluoride sensoras described previously.

Since the degree of DFPase modification could not be determineddirectly, the reaction of Desmodur N3400 and enzyme Lysine residues wasmimicked using Bradykinin potentiator B, a low molecular weight peptide(1182.4 Da) containing one Lysine residue. The extent of Lysinemodification was determined using MALDI-TOF for various reaction time(15 min to 17 hr).

MALDI-MS analyses were performed with a Perseptive Biosystems Voyagerelite MALDI-TOF. The acceleration voltage was set to 20 kV in a linearmode. The PEGylated enzyme solution (1-2 mg/ml) was mixed with an equalvolume of the matrix solution (0.5 ml water, 0.5 ml acetonitrile, 2 μlTFA and 8 mg α-cyano-4-hydroxycinnamic acid), and 2 μl of the finalsolution was spotted on the plate target. Spectra were recorded afterevaporation of the solvent mixture, and were calibrated externally withFMRP and ACTH.

DFPase modified with Desmodur N3400 was further immobilized intopolyurethane coatings as described previously.

ECC Thermostability

Native and immobilized DFPase were added to buffer (10 mM BTP, 5 mMCaCl₂, pH 7.5) incubated at 65° C., and assayed at room temperature inbuffered media (10 mM BTP, 5 mM CaCl₂, pH 7.5) as described above.

The thermostability of dry ECC's was determined at room temperature.After fixed periods of storage under ambient conditions, the ECC sampleswere assayed for activity at room temperature in buffered media (10 mMBTP, 5 mM CaCl₂, pH 7.5) as described above.

Results and Discussion

Reversibility of DFPase Attachment to ECC's

The extent to which DFPase is irreversibly attached to the polymer wasdetermined using the Bradford reagent. DFPase-containing polyurethanecoatings were peeled from panels, cut into small pieces, and extensivelyrinsed with distilled water. Less than 4% (w/w) of the protein loaded tothe ECC was detected in the rinsates, indicating that the immobilizationefficiency approached 100%.

Enzyme Distribution in ECC's

When enzymes are incorporated into films, a key issue is whether theenzyme is equally distributed in the film. Gold labeling has been usedto localize immobilized enzyme in polyurethane monolith foams in theprior art. Therefore, in the present invention DFPase was localized inECC's via conjugation to colloidal gold particles.

FIG. 2 illustrates an enzyme distribution in polyurethane coating.Gold/DFPase-containing coatings were analyzed using dark field (A;0.0007 mg_(gold)/g_(coating)) and inverse (negative) images taken usinglight microscopy (B; 0.0116 mg_(gold)/g_(coating)). Negative images wereused in this case because the thickness of the coating and the highconcentration of gold particles made it difficult to obtain focusedimages. Cross sections of the coatings were obtained using Transmissionelectron microscopy (C and D). The arrows with filled heads show some ofthe gold/enzyme particles, while the arrows with emptied heads show someof the gold/enzyme conjugate clusters. The arrowheads indicate theextremities of coating samples within the embedded resin. The starsdesignate some unfocussed areas as a result of high gold particleconcentration and uneven surface. Bubbles in the coating are indicatedby the letter h. Size bar shown in B represent panels A and B. Size barin C and D indicate sizes in those panels.

FIGS. 2A and 2B are micrographs of gold/DFPase conjugate-containingcoatings obtained by dark field microscopy (0.001 mg_(gold)/g_(coating))and inverse image light microscopy (0.012 mg_(gold)/g_(coating)),respectively. As the concentration of immobilized colloidal gold/enzymeconjugate is increased by 12-fold it becomes apparent that theimmobilized gold/enzyme complexes are uniformly distributed within thecoating. The TEM's of the cross section of gold/enzyme-containingcoating (0.012 mg_(gold)/g_(coating)) are given in FIGS. 2C (originally2500-fold enlargement) and 2D (10,000-fold magnification). Similarly tolight microscopy, TEM shows that the gold/enzyme particles and clustersare randomly distributed at the microscale level. This implies that thesynthesis of gold/DFPase conjugate-containing coating leads to thehomogeneous immobilization of gold/DFPase complexes in the polymericmatrix. By extrapolation one can predict that the DFPase localconcentration in a film should not be location dependent.

Activity of ECC's

ECC's were prepared using the polyisocyanate prepolymers XP-7007,XP-7148 and XP-7063. FIG. 3 shows the activity of each ECC as a functionof initial DFPase loading. The activity is directly proportional to theenzyme concentration, which implies that there is no significant masstransfer limitation. Since FIG. 2 indicates that the films arenon-porous, this result implies (as we will discuss in detail later)that only enzyme in a thin external layer of the film is accessible tosubstrate.

The hydrophilicity of polyisocyanate decreases in the orderXP-7148>XP-7063>XP-7007. Interestingly, the apparent activity retentionof ECC's increases as the hydrophilicity of polyisocyanate decreases(See FIG. 3). Studies of enzyme activity in dehydrated organic solventsdemonstrate that enzymes prefer hydrophobic environments. It may not becoincidental that less hydrophilic polyisocyanates are superior ECCmaterials. With respect to FIG. 3, coatings were synthesized with polyolXP-7093 and polyisocyanate XP-7007 (Closed diamond), XP-7063 (closedcircles) and XP-7148 (closed squares) in buffered solution (10 mMbis-tris-propane, 5 nM CaCl₂) at pH 7.5. The closed triangles correspondto the apparent activity of coatings synthesized starting from DFPasemodified with Desmodur N3400, polyol XP-7093 and polyisocyanate XP-7007.The activity of the bioplastic is reported at a 3 mM DFP concentration.

The use of polyisocyanate XP-7007 generated ECC's with the highestlevels of apparent activity retention, and thus subsequent environmentswere preformed with XP 7007-containing-ECC's. The apparent kineticcharacteristics calculated by assuming all the loaded enzyme isavailable (Table 1, Experiment 1^(b*)) lead to an observable activityretention (11%) rather than intrinsic retention.

Effective Diffusivity of DFP in ECC, D_(eff)

To understand activity retention in ECC's the diffusivity of thesubstrate in the film was assessed. Using Equation 1, D_(eff) was foundto be (5+/−1)×10⁻¹⁰ m²/min. With reference to FIG. 4, coatings weresynthesized using the polyol XP-7093 and polyisocyanate XP-7007, and theexperiment was conducted in buffered medium (10 mM bis-tris-propane, 5mM CaCl₂) at pH 7.5 by means of a cell diffusion apparatus. It is knownthat D_(eff) is two to three orders of magnitude lower than thediffusion coefficients of gases into liquids or organic solutes intohydrogels. Similarly, in the prior art high resistance of two-componentwaterborne polyurethane coatings to diffusion of chloride ions wasobserved. The accessibility of enzyme located within the coating tosubstrate is clearly limited by the low coating permeability. Onceagain, this result indicates that the degree of penetration of DFP intocoating should be taken into account in order to determine the activityretention of ECC's.

FIG. 5 shows the profile for DFP concentration in donor and receptorcell over time when using a DFPase-ECC (3.6 mg/g_(coating)), and aninitial concentration of 4 mM DFP in both cells. The experiments wereconducted in buffered medium (10 mM bis-tris-propane, 5 mM CaCl₂) at pH7.5 using an initial DFP concentration of 4 mM in both donor andreceptor cells. The DFP concentrations in donor (closed diamonds) andreceptor (closed circles) cells were determined over time. Thetheoretical DFP profiles in the donor and receptor cells are identicaldue to symmetry. The experimental concentration curves in donor andreceptor cells are, thus, described by the same simulated profile(dashed line) using Equations 2-7 and Athena Visual 7.1.1. The profilesfor the decrease in DFP concentration in donor and receptor cells followsimilar trends. Assuming immobilized DFPase is homogenously distributedin the coating (as implied in FIG. 2), the enzymatic activity retentionis therefore almost the same on both sides of coating. During curing,the ECC upper and lower surfaces are in contact with the TPO panel andexposed to air, respectively. As given by the little difference inactivity retention of the ECC's external surfaces, the air interface andthe polymeric/hydrophobic environment do not influence the ECC activityretention.

DFP concentration profiles in the donor and receptor cells were alsomeasured for a DFPase-ECC (3.6 mg/g_(coating)) with no DFP in thereceptor cell (See FIG. 6). Coatings were synthesized using the polyolXP-7093 and polyisocyanate XP-7007, and a DFPase loading of 3.6mg/g_(coating). The experiments were conducted in buffered medium (10 mMbis-tris-propane, 5 mM CaCl₂) at pH 7.5 starting with DFP (4 mM) in thedonor cell and no DFP in the receptor cell. FIG. 6 a shows the DFPconcentrations in donor (closed diamonds) and receptor (closed circles)cells were measured over time. The simulated DFP concentration profilesin donor (dash line) and receptor (dotted line) cells were determinedusing Equation 2-7 and Athena Visual 7.1.1. FIG. 6 b shows the substrateconcentration profile in the ECC's was calculated at 0 (medium dashedline), 30 (solid line), 60 (small dashed line), 90 (dashed-dotted line),120 (dotted line), 180 (dashed-dotted-dotted line) and 280 (long dashline). Equation 3 describes well the experimental results (FIG. 6 a).The estimated intrinsic Michaelis constant of immobilized DFPase,K_(M,int) (Table 1, Experiment 2^(b**)), is similar to that obtainedwithout the diffusion apparatus (Table 1, Experiment 1^(b*)).Interestingly, by taking into account the coating resistance tosubstrate diffusion, k_(cat,int) (Table 1, Experiment 2^(b**)) was foundto be 2.4 times higher than the apparent k_(cat,app) measured withoutthe diffusion apparatus (Table 1, Experiment 1^(b*)). As shown by thesimulated substrate profiles within the coating at differentexperimental times (FIG. 6 b), the substrate penetrates a third of thecoating over the time course of the experiment. Clearly, the estimationof apparent kinetic parameters involves solely the degradation of DFP ina layer of immobilized enzyme at the coating surface. Consequently, theapparent enzymatic efficiency of DFPase-ECC's is based on the activityretention of this external layer of immobilized DFPase. As given by theintrinsic kinetic constants of DFPase-ECC, the intrinsic activityretention within this layer is 38%. The ratio of apparent to intrinsic$k_{cat},{R = {\frac{k_{{cat},{app}}}{k_{{cat},{int}}} = 0.4}},$gives the proportion of immobilized DFPase in ECC's reachable by thesubstrate during activity measurements without the diffusion apparatus.Desmodur N3400 Polyisocyanate-Modified ECC's

The vigorous mixing of Bradykinin potentiator B-containing aqueoussolution with Desmodur N3400 polyisocyanate ensured the chemicalmodification of the peptide Lysine residue with the dimer of HDI, asobserved using MALDI-TOF. A reaction yield fluctuating between 70 and90% was reached for a 15 min reaction time, and was not increased byfurther mixing of the peptide solution with the Desmodur N3400polyisocyanate phase.

Polyisocyanate Desmodur N3400 is based on the uretdione of HDI which isknown to migrate from the bulk to the polymer/air interface duringcoating curing. By modifying DFPase with Desmodur N3400 prior to itsimmobilization into coatings, it was expected that the immobilizedenzyme would be mainly concentrated within an external layer at thecoating surface. Consequently, immobilized DFPase would be wellaccessible to substrate, leading to an increased apparent activityretention. Given the fast favorable reaction between isocyanates of thedimer of HDI and the Lysine residue of Bradykinin potentiator B, DFPasewas reacted with Desmodur N3400 for 15 min. No loss of enzymaticactivity was observed. As shown in FIG. 3 and Table 1 (Experiment1b^(#*)), the pre-treatment of DFPase with Desmodur N3400 produced a 64%increase in apparent efficiency of ECC's.

Thermostability of ECC's

As explained in the previous section, not all of the immobilized enzymeis seen by the substrate during activity measurement. Since theinaccessible enzyme does not interfere with the rate determinations thethermal stability of the film can be determined without specialconsideration of diffusion resistances.

Unlike native DFPase, immobilized DFPase has a biphasicthermoinactivation profile at 65° C. (See FIG. 7). Deactivation ofimmobilized DFPase (closed squares) and native DFPase (closed diamonds)were conducted in buffered solution (10 mM BTP, 5 mM CaCl₂, pH 7.5). Theremaining enzymatic activity was measured over time at room temperaturein buffered media (10 mM BTP, 5 mM CaCl₂, pH 7.5) using DFP (3 mM) as asubstrate. The biphasic behavior was described with a four parametermodel, and the kinetic constants α₁ (0.34±0.03), α₂ (0.10±0.01), k₁(1.3±0.1) and k₂ (0.042±0.003), were determined using the algorithm ofMarquardt-Levenberg (SigmaPlot Version 2.0). An elevated temperature of65° C. was used to inactivate the enzyme in order to perform experimentson a reasonable time scale. For this range of incubation periods, thetwo component polyurethane coatings did not dissolve significantly intothe aqueous phase. Initially, the ECC follows a deactivation trendsimilar to that for native enzyme. This initial rapid deactivationleads, however, to the formation of a stable and active form ofimmobilized enzyme with a 6-7% residual activity. No significant changein the activity of the highly stable form of the DFPase-ECC is observedover 350 min. The biphasic deactivation kinetics of the ECC can bemodeled by a four-parameter model, which assumes the following scheme:$\begin{matrix}{E\overset{k_{1}}{\longrightarrow}{\overset{\alpha_{1}}{E}}_{1}\overset{k_{2}}{\longrightarrow}{\overset{\alpha_{2}}{E}}_{2}} & {{Equation}\quad 9}\end{matrix}$E, E₁ and E₂ correspond to the initial, intermediate and final state ofenzyme. α₁ and α₂ are the residual activities of E₁ and E₂,respectively, while k₁ and k₂ represent first-order deactivation rates.The analytical solution for the enzymatic activity, α, is given byEquation 10. $\begin{matrix}{a = {{\left( {1 + \left( \frac{{\alpha_{1}k_{1}} - {\alpha_{2}k_{2}}}{k_{2} - k_{1}} \right)} \right){\exp\left( {{- k_{1}}t} \right)}} + {\left( \frac{k_{1}\left( {\alpha_{2} - \alpha_{1}} \right)}{k_{2} - k_{1}} \right){\exp\left( {{- k_{2}}t} \right)}} + \alpha_{2}}} & {{Equation}\quad 10}\end{matrix}$Where t represents the time of deactivation. The fit of the data toEquation 10 are given in FIG. 7.

Another kinetic model assuming the existence of two different forms ofDFPase in ECC's with different deactivation pathways, and requiring onlyfour physical parameters did not adequately describe the experimentaldata. Further more complex mechanisms were not considered as theyinvolved five or more parameters.

The immobilization of DFPase in polyurethane foam and PEGylation alsoinduced a transition from first order to biphasic inactivation kinetics.We believe that thermoinactivation of the DFPase-ECC results fromstructural changes similar to those described previously for thethermoactivation of DFPase-containing polyurethane foam monoliths.

DFPase-ECC's exhibit a higher stability at room temperature than at 65°C. Indeed, DFPase-ECC's lose only 40% activity after 100 days of storageat room temperature (See FIG. 8). The remaining enzymatic activity wasmeasured over time in a buffered media (10 mM BTP, 5 mM CaCl₂, P.H. 7.5)using FP (mM as a substrate. Given the high stability of ECC'smaintained dry under ambient conditions, the resulting catalyst shouldbe an effective decontaminant for a variety of applications.

Therefore, covalent incorporation of DFPase into waterborne polyurethanecoatings has been performed in the present invention in a single stepprotein-polymer synthesis using polyol and polyisocyanates. The use ofpolyisocyanate XP-7007 and enzyme modified with Desmodur N3400 duringthe immobilization process leads to the highest intrinsic catalyticefficiency (with 18 to 38% activity retention). At high temperature,DFPase-ECC's lose 93% of their activity quickly, but then becomehyper-stable.

While the present invention has been described in conjunction withpreferred embodiments thereof, those of ordinary skill will recognizethat many modifications and variations thereof are possible. Theforegoing description and the following claims are intended to cover allsuch modifications and variations. TABLE 1 Kinetic parameters forDFPase- containing coatings and soluble DFPase K_(M) k_(cat)k_(cat)/K_(M) Experiment (mM) (s⁻¹) (s⁻¹· mM⁻¹) 1^(a*); intrinsic nativeDFPase 0.79 ÷ 0.02 232 ÷ 2  293 ÷ 0.3 1^(b*); apparent ECC 1.3 ÷ 0.2 43÷ 3 33 ÷ 7  1^(b#*); apparent ECC 1.3 ÷ 0.2 70 ÷ 6  54 ÷ 13 2^(b#);intrinsic ECC 1.0 ÷ 0.1 211 ÷ 8  211 ÷ 4   The errors on specificconstants were calculated as follows:${\Delta\left( \frac{k_{cat}}{K_{M}} \right)} = {\left( \frac{k_{cat}}{K_{M}} \right) \cdot \left\lbrack {\frac{\Delta\quad k_{cat}}{k_{cat}} + \frac{\Delta\quad K_{M}}{K_{M}}} \right\rbrack}$^(a): native DFPase ^(b): polyurethane coatings ^(*): The kineticparameters were evaluated at room temperature in buffered media (10mMbis-tris-propane,5 mM4 CaCl_(2, pH 7.5) using substrate) concentrationsvarying from 0 to 20 mM and fluoride ion electrode, by applying theMichaelis-Menten equation as a model and using a non-linear regression(SigmaPlot Version 2). ^(#): DFPase was modified with Desmodur N3400polyisocyanate prior to immobilization into polyurethane coatings.^(**): The kinetic parameters were evaluated at room temperature inbuffered media (10mM bis-tris-propane, 5 mM CaCl₂, pH 7.5) using thediffusion cell apparatus.

1. An enzyme-containing polyurethane made by the process comprising thesteps of: (a) reacting a mixture of (i) a polyol dispersion coreactantand (ii) an enzyme to create an aqueous mixture; (b) adding awater-dispersible polyisocyanate based on hexamethylene diisocyanate tothe aqueous mixture (c) allowing the polyisocyanate and aqueous mixtureto form a dispersion; (d) applying the dispersion onto a substrate toproduce an enzyme-containing polyurethane; and (e) curing theenzyme-containing polyurethane.
 2. The enzyme-containing polyurethanemade by the process of claim 1 in which the amount of polyol dispersionto which polyisocyanate is added in step (b) is approximately 2 timesgreater than the amount of the polyisocyanate.
 3. The enzyme-containingpolyurethane made by the process of claim 2 in which the polyoldispersion used in step (a) has a water content of approximately 70 wt.%.
 4. The enzyme-containing polyurethane made by the process of claim 3in which a polyethylene modified polydimethyl siloxane surfactant ispresent during step (a).
 5. The enzyme-containing polyurethane made bythe process of claim 4 in which a bis-tris propane buffer and CaCl₂ arepresent during step (a).
 6. The enzyme-containing polyurethane made bythe process of claim 5 in which the polyol dispersion coreactant ispresent during step (c) in an amount which is 2.5 times that of thepolyisocyanate present in step (c).
 7. The enzyme-containingpolyurethane made by the process of claim 6 in which approximately0.02-9 mg diisopropyflurophosphatase are used in step (a).
 8. Theenzyme-containing polyurethane made by the process of claim 1 havingbetween 10% and 100% activity retention.
 9. The enzyme-containingcoating made by the process of claim 1 having a degree of immobilizationof the diisopolyflurophosphatase of approximately 100%.